A promoter directing α-amanitin-sensitive transcription of GARP ,the major surface antigen of insect stage Trypanosoma congolense

Abstract

The major surface antigen of procyclic and epimastigote forms of Trypanosoma congolense in the tsetse fly is GARP (glutamic acid/alanine-rich protein) ,which is thought to be the analogue of procyclin/PARP in Trypanosoma brucei. We have studied two T.congolense GARP loci (the 4.3 and 4.4 loci) whose transcription is α-amanitin sensitive. Whilst a transcriptional gap 5′ of the first GARP gene in the cloned region of the 4.4 locus could not be detected ,such a gap was present in the 5′ flank of the first GARP gene in the 4.3 locus. We have located a GARP transcription start site and ,using reporter gene constructs containing a putative GARP promoter region in transient transfection studies ,we have demonstrated promoter activity for the test region in T.congolense. There are species-specific differences in sequences regulating expression of the two major surface antigens ,GARP and procyclin/PARP: the GARP promoter is inactive in T.brucei while the procyclin/PARP promoter is inactive in T.congolense. We have defined the splice acceptor site for the 4.3 GARP gene by sequencing and by 5′ RT-PCR and demonstrated microheterogeneity in GARP polyadenylation by 3′ RT-PCR. It appears that some GARP and procyclin/PARP RNA processing signals ,although similar ,are also species-specific.